呼吸道合胞病毒血清中和抗体检测方法的建立

Development of a detection method for serum neutralizing antibody against respiratory syncytial virus

目的 建立荧光检测法测定呼吸道合胞病毒(respiratory syncytial virus, RSV)血清中和抗体的检测方法,并对其进行验证和初步应用。方法 采用反向遗传学拯救的表达绿色荧光蛋白的重组呼吸道合胞病毒(recombinant RSV experssed the enhanced green fluorescent protein, RSV-EGFP)进行RSV重组腺病毒疫苗免疫后血清的检测。通过对RSV-EGFP在人喉癌上皮(human epithelioma-2,HEp-2)细胞上荧光的检测波长、检测时间、病毒的感染剂量和HEp-2的细胞密度进行优化,确定合适的条件后,再将RSV血清倍比系列稀释后与RSV-EGFP混合接种至HEp-2细胞培养,利用多功能酶标仪检测HEp-2细胞中表达的绿色荧光蛋白的荧光强度,从而计算其中和抗体滴度。同时,通过对RSV重组腺病毒载体疫苗免疫棉鼠血清的检测进一步评估中和抗体检测方法的精密度、重复性、准确度以及边缘孔效应。结果 结果显示,当检测的激发波长为479 nm,发射波长为517 nm,检测时间为病毒感染后48 h,最佳病毒感染剂量为3 000 pfu/孔,检测的最佳细胞密度为2.0×10~4个/孔时,相对荧光强度最高,且病毒数量与荧光强度之间呈现出较好的剂量-效应关系。采用该方法检测不同抗体滴度的血清样本,日内与日间精密度的CV均<15%;同一血清样本10次重复性检测结果最大值与最小值的比值均≤4,且RSD均<15%;用建立的方法实际测得的中和滴度结果与稀释后理论中和滴度(酶联免疫斑点法)的回收率均在80%~120%之间;在边缘孔与非边缘孔检测RSV血清中和抗体滴度结果一致性较好(P>0.05)。结论 建立的荧光检测法较酶联免疫斑点法操作简便、快捷,可被用来检测RSV血清中和抗体效价。

Objective To establish and validate a fluorescence assay for the determination of serum neutralizing antibody to respiratory syncytial virus(RSV). Methods A reverse genetics-rescued recombinant RSV expressing the enhanced green fluorescent protein(recombinant RSV expressed the enhanced green fluorescent protein, RSV-EGFP) was used for the detection of serum after immunization with RSV recombinant adenovirus vaccine. The detection wavelength, detection time, infectious dose of virus and cell density of human epithelial laryngeal carcinoma-2(HEp-2) were optimized for RSV-EGFP fluorescence on Hep-2 cells, and the appropriate conditions were determined, then the RSV serum was diluted in a multiplicity series and mixed with RSV-EGFP for inoculation into HEp-2 cell culture. The fluorescence intensity of the enhanced green fluorescent protein expressed in HEp-2 cells was detected using a multifunctional enzyme labeler, so as to calculate its neutralizing antibody titer. The precision, reproducibility and accuracy of the neutralizing antibody assay were further evaluated by testing the serum of cotton mice immunized with RSV recombinant adenovirus vaccine. as well as the edge hole effect. Results The results showed that the relative fluorescence intensity was highest when the excitation wavelength of the assay was 479 nm, the emission wavelength was 517 nm, the assay time was 48 h after viral infection, the optimal viral infection dose was 3 000 pfu/well, and the optimal cell density of the assay was 2.0×10~4 wells/well, and a good dose-effect relationship between the viral counts and the fluorescence intensities was demonstrated. The CV of intra-day and inter-day precision of serum samples with different antibody titers detected by the method were all <15%;the ratios of maximum to minimum of 10 repetitive assay results of the same serum sample were all ≤4,and the RSDs were all <15%;the recoveries of the actual neutralization titer results measured by the established method and the theoretical neutralization titer(Immuno-plaque assay) after dilution were all in the range of 80% to 120%;the consistency of the RSV serum neutralizing antibody titer results detected in marginal and non-marginal wells was good(P>0.05). Conclusion The established fluorescence assay is simpler and faster than the Immuno-plaque assay, and can be used to detect RSV serum neutralizing antibody potency.